Deuterated rapamycin compounds, method and uses thereof

ABSTRACT

The synthesis of deuterated analogues of rapamycin is disclosed together with a method for use for inducing immunosupression and in the treatment of transplantation rejection, graft vs host disease, autoimmune diseases, diseases of inflammation leukemia/lymphoma, solid tumors, fungal infections, hyperproliferative vascular disorders. Also described is a method for the synthesis of water soluble deuteratred rapamycin compounds and their use as described above.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/148,623, which is based on provisional patentapplication No. 60/057,632, both of which are relied on and incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to deuterated derivatives of rapamycin anda method for using them in the treatment of transplantation rejection,host vs. graft disease, graft vs. host disease, leukemia/lymphomahyperproliferative vascular disorders, autoimmune diseases, diseases ofinflammation, solid tumors, and fungal infections.

[0003] Rapamycin, known as sirolimusis, is a 31-membered macrolidelactone, C₅₁H₇₉NO₁₃, with a molecular mass of 913.6 Da. In solution,sirolimus forms two conformational trans-, cis-isomers with a ratio of4:1 (chloroform) due to hindered rotation around the pipecolic acidamide bond. It is sparingly soluble in water, aliphatic hydrocarbons anddiethyl ether, whereas it is soluble in alcohols, halogenatedhydrocarbons and dimethyl sulfoxide. Rapamycin is unstable in solutionand degrades in plasma and low-, and neuteral -pH buffers at 37° C. withhalf-life of <10 h. the structures of the degradation products haverecently been characterized. Rapamycin is a macrocyclic trieneantibiotic produced by Streptomyces hygroscopicus, which was found tohave antifungal activity, particularly against Candida albicans, both invitro and in-vivo [C. Vezina et al., J. Antibiot. 28, 721 (1975); S. N.Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J.Antibiot. 31, 539 (1978); U.S. Pat. No. 3,929,992; and U.S. Pat. No.3,993,749].

[0004] Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)]disclosed that rapamycin is effective in the experimental allergicencephalomyelitis model, a model for multiple sclerosis; in the adjuvantarthritis model, a model for rheumatoid arthritis; and effectivelyinhibited the formation of IgE-like antibodies.

[0005] The immunosuppressive effects of rapamycin have been disclosed inFASEB 3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclicmolecules, also have been shown to be effective as immunosuppressiveagents, therefore useful in preventing transplant rejection [FASEB 3,3411 (1989); FASEB 3, 5256 (1989); and R. Y. Calne et al., Lancet 1183(1978)]. Although it shares structural homology with theimmunosuppressant tacrolimus and binds to the same intracellular bindingprotein in lymphocytes, rapamycin inhibits S6p70-kinase and thereforehas a mechanism of immunosuppressive action distinct from that oftacrolimus. Rapamycin was found to prolong graft survival of differenttransplants in several species alone or in combination with otherimmunosupressants. In animal models its spectrum of toxic effects isdifferent from that of cyclosporin or FK-506., comprising impairment ofglucose homeostasis, stomach, ulceration, weight loss andthrombocytopenia, although no nephrotoxicity has been detected.

[0006] Mono- and diacylated derivatives of rapamycin (esterified at the28 and 43 positions) have been shown to be useful as antifungal agents(U.S. Pat. No. 4,316,885) and used to make water soluble prodrugs ofrapamycin (U.S. Pat. No. 4,650,803). Recently, the numbering conventionfor rapamycin has been changed; therefore according to ChemicalAbstracts nomenclature, the esters described above would be at the 31-and 42-positions. Carboxylic acid esters (PCT application No. WO92/05179), carbamates (U.S. Pat. No. 5,118,678), amide esters (U.S. Pat.No. 5,118,678), (U.S. Pat. No. 5,118,678) fluorinated esters (U.S. Pat.No. 5,100,883), acetals (U.S. Pat. No. 5,151,413), silyl ethers (U.S.Pat. No. 5,120,842), bicyclic derivatives (U.S. Pat. No. 5,120,725),rapamycin dimers (U.S. Pat. No. 5,120,727) and O-aryl, O-alkyl,O-a-lkyenyl and O-alkynyl derivatives (U.S. Pat. No. 5,258,389) havebeen described.

[0007] Rapamycin is metabolized by cytochrome P-450 3A to at least sixmetabolites. During incubation with human liver and small intestinalmicrosomes, sirolimus was hydroxylated and demethylated and thestructure of 39-O-demethyl sirolimus was identified. In bile ofsirolimus-treated rats >16 hydroxylated and demethylated metaboliteswere detected.

[0008] In rapamycin, demethylation of methoxy group at C-7 Carbon willlead to the change in the conformation of the Rapamycin due to theinteraction of the released C-7 hydroxyl group with the neighbouringpyran ring system which is in equilibrium with the open form of the ringsystem. The C-7 hydroxyl group will also interact with the triene systemand possibly alter the immunosupressive activity of rapamycin. Thisaccounts for the degradation of rapamycin molecule and its alteredactivity.

[0009] Stable isotopes (e.g., deuterium, ¹³C, ¹⁵N, ¹⁸O) arenonradioactive isotopes which contain one additional neutron than thenormally abundant isotope of the atom in question. Deuterated compoundshave been used in pharmaceutical research to investigate the in vivometabolic fate of the compounds by evaluation of the mechanism of actionand metabolic-pathway of the non deuterated parent compound. (Blake etal. J. Pharm. Sci. 64, 3, 367-391,1975). Such metabolic studies areimportant in the design of safe, effective therapeutic drugs, eitherbecause the in vivo active compound administered to the patient orbecause the metabolites produced from the parent compound prove to betoxic or carcinogenic (Foster et al., Advances in drug Research Vol. 14,pp. 2-36, Academic press, London, 1985).

[0010] Incorporation of a heavy atom particularly substitution ofdeuterium for hydrogen, can give rise to an isotope effect that canalter the pharmacokinetics of the drug. This effect is usuallyinsignificant if the label is placed in a molecule at the metabolicallyinert position of the molecule.

[0011] Stable isotope labeling of a drug can alter its physico-chemicalproperties such as pKa and lipid solubility. These changes may influencethe fate of the drug at different steps along its passage through thebody. Absorption, distribution, metabolism or excretion can be changed.Absorption and distribution are processes that depend primarily on themolecular size and the lipophilicity of the substance.

[0012] Drug metabolism can give rise to large isotopic effect if thebreaking of a chemical bond to a deuterium atom is the rate limitingstep in the process. While some of the physical properties of a stableisotope-labeled molecule are different from those of the unlabeled one,the chemical and biological properties are the same, with one importantexception: because of the increased mass of the heavy isotope, any bondinvolving the heavy isotope and another atom will be stronger than thesame bond between the light isotope and that atom. In any reaction inwhich the breaking of this bond is the rate limiting step, the reactionwill proceed slower for the molecule with the heavy isotope due tokinetic isotope effect. A reaction involving breaking a C-D bond can beup to 700 per cent slower than a similar reaction involving breaking aC—H bond.

[0013] More caution has to be observed when using deuterium labeleddrugs. If the C-D bond is not involved in any of the steps leading tothe metabolite, there may not be any effect to alter the behavior of thedrug. If a deuterium is placed at a site involved in the metabolism of adrug, an isotope effect will be observed only if breaking of the C-Dbond is the rate limiting step. There are evidences to suggest thatwhenever cleavage of an aliphatic C—H bond occurs, usually by oxidationcatalyzed by a mixed-function oxidase, replacement of the hydrogen bydeuterium will lead to observable isotope effect. It is also importantto understand that the incorporation of deuterium at the site ofmetabolism slows its rate to the point where another metabolite producedby attack at a carbon atom not substituted by deuterium becomes themajor pathway by a process called “metabolic switching”.

[0014] It is also observed that one of the most important metabolicpathways of compounds containing aromatic systems is hydroxylationleading to a phenolic group in the 3 or 4 position to carbonsubstituents. Although this pathway involves cleavage of the C—H bond,it is often not accompanied by an isotope effect, because the cleavageof this bond is mostly not involved in the rate-limiting step. Thesubstitution of hydrogen by deuterium at the stereo center will induce agreater effect on the activity of the drug.

[0015] Clinically relevant questions include the toxicity of the drugand its metabolite derivatives, the changes in distribution orelimination (enzyme induction), lipophilicity which will have an effecton absorption of the drug. Replacement of hydrogen by deuterium at thesite involving the metabolic reaction will lead to increased toxicity ofthe drug. Replacement of hydrogen by deuterium at the aliphatic carbonswill have an isotopic effect to a larger extent. Deuterium placed at anaromatic carbon atom, which will be the site of hydroxylation, may leadto an observable isotope effect, although this is less often the casethan with aliphatic carbons. But in few cases such as in penicillin, thesubstitution on the aromatic ring will induce the restriction ofrotation of the ring around the C—C bond leading to a favorablestereo-specific situation to enhance the activity of the drug.

[0016] Approaching half a century of stable-isotope usage in humanmetabolic studies has been without documented significant adverseeffect. Side-effects with acute D dosing are transitory with nodemonstrated evidence of permanent deleterious action. The threshold ofD toxicity has been defined in animals and is far in excess ofconcentrations conceivably used in human studies (Jones P J, LeatherdaleST Clin Sci (Colch) 1991 April;80(4):277-280). The possibility that Dmay have additional beneficial pharmacological applications cannot beexcluded. For isotopes other than D, evidence of observed toxicityremains to be produced even at dosages far in excess of the range usedin metabolic studies. Absence of adverse effect may be attributable tosmall mass differences and the similar properties of tracer andpredominantly abundant isotopes. The precision of extrapolating toxicitythresholds from animal studies remains unknown. However, shouldperturbation of the delicate homoeostatic characteristic of livingorganisms occur with use of stable isotopes, it is almost undoubtedly atsome level of administration greatly in excess of those administeredcurrently in biomedical research.

[0017] In the prior art, no details are described regarding deuteratedderivatives to improve the stability of rapamycin molecule and alsoabout glycosylated deuterated rapamycin to improve the stability andalso the solubility of the molecule in order to increase thebio-availability of the drug. We therefore defined the global objectiveof preparing a rapamycin derivative which is more stable, less prone todegradation, and more water soluble to improve the bioavailability.

SUMMARY OF THE INVENTION

[0018] Deuteration of the rapamycin molecule results in alteredphysicochemical and pharmacokinetic properties which enhance itsusefulness in the treatment of transplantation rejection, host vs. graftdisease, graft vs. host disease, leukemia/lymphoma, hyperproliferativevascular disorders, autoimmune diseases, diseases of inflammation, solidtumors, and fungal infections.

[0019] Deuterium isotope is selected based on the fact that if ¹³C, ¹⁵Nor another heavy isotope differing from the light one by less than 10%in mass is incorporated at the site of metabolism, there may be a smallisotope-effect. In addition to this, there are secondary isotope effectsaway from the site of isotope substitution due to changes in electronicenvironment.

[0020] Substitution of deuterium in methyl groups of rapamycin willresult in a slower rate of oxidation of the C-D bond relative to therate of oxidation of a non deuterium substituted C—H bond. The isotopiceffect acts to reduce formation of demethylated metabolites and therebyalters the pharmacokinetic parameters of the drug. Lower rates ofoxidation, metabolism and clearance result in greater and more sustainedbiological activity. Deuteration is targeted at various sites of therapamycin molecule to increase the potency of drug, reduce toxicity ofthe drug, reduce the clearance of the pharmacologically active moietyand improve the stability of the molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is the chemical structure of 7-deuteromethyl rapamycinshowing sites of deuteration.

[0022]FIG. 2 is the chemical structure of epi-7 deuteromethyl rapamycinshowing sites of deuteration.

[0023]FIG. 3 is the chemical structure of 7,43-d₆-rapamycin showingsites of deuteration.

[0024]FIG. 4 is the chemical structure of 31,42-d₂ showing sites ofdeuteration.

[0025]FIG. 5 illustrates the preparation of glycosylateddeuterorapamycin.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Substitution of deuterium for ordinary hydrogen and deuteratedsubstrates for protio metabolites can produce profound changes inbiosystems. Isotopically altered drugs have shown widely divergentpharmacological effects. Pettersen et al., found increased anti-cancereffect with deuterated 5,6-benzylidene-dl-L-ascorbic acid (Zilascorb)[Anticancer Res. 12, 33 (1992)].

[0027] Substitution of deuterium in methyl groups of rapamycin willresult in a slower rate of oxidation of the C-D bond relative to therate of oxidation-of a non deuterium substituted C—H bond. The isotopiceffect acts to reduce formation of demethylated metabolites and therebyalters the pharmacokinetic parameters of the drug. Lower rates ofoxidation, metabolism and clearance result in greater and more sustainedbiological activity. Deuteration is targeted at various sites of therapamycin molecule to increase the potency of drug, reduce toxicity ofthe drug, reduce the clearance of the pharmacologically active moietyand improve the stability of the molecule.

[0028] Determination of the physicochemical, toxicological andpharmacokinetic properties can be made using standard chemical andbiological assays and through the use of mathematical modelingtechniques which are known in the chemical andpharmacological/toxicological arts. The therapeutic utility and dosingregimen can be extrapolated from the results of such techniques andthrough the use of appropriate pharmacokinetic and/or pharmacodynamicmodels.

[0029] The compounds of this invention may be administered neat or witha pharmaceutical carrier to an animal, such as a warm blooded mammal,and especially humans, in need thereof. The pharmaceutically effectivecarrier may be solid or liquid.

[0030] A solid carrier can include one or more substances which may alsoact as flavoring agents, lubricants, solubilizers, suspending agents,fillers, glidants, compression aids, binders or tablet-disintegratingagents; it can also be an encapsulating material. In powders, thecarrier is a finely divided solid which is in admixture with the finelydivided active ingredient. In tablets, the active ingredient is mixedwith a carrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets may contain up to 99% of the active ingredient. Suitable solidcarriers include, for example, calcium phosphate, magnesium stearate,talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, lowmelting waxes and ion exchange resins.

[0031] Liquid carriers are used in preparing solutions, suspensions,emulsions, syrups, elixirs and pressurized compositions. The activeingredient can be dissolved or suspended in a pharmaceuticallyacceptable liquid carrier such as water, an organic solvent, a mixtureof both or pharmaceutically acceptable oils or fats. The liquid carriercan contain other suitable pharmaceutical additives such assolubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoringagents, suspending agents, thickening agents, colors, viscosityregulators, stabilizers or osmo-regulators. Suitable examples of liquidcarriers for oral and parenteral administration include water (partiallycontaining additives as above, e.g. cellulose derivatives, possiblysodium carboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

[0032] Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

[0033] The pharmaceutical composition can be in unit dosage form, e.g.as tablets or capsules. In such form, the composition is sub-divided inunit dose containing appropriate quantities of the active ingredient;the unit dosage forms can be packaged compositions, for example,packeted powders, vials, ampoules, prefilled syringes or sachetscontaining liquids. The unit dosage form can be, for example, a capsuleor tablet itself, or it can be the appropriate number of any suchcompositions in package form. The dosage to be used in the treatmentmust be subjectively determined by the attending physician.

[0034] In addition, the compounds of this invention may be employed as asolution, cream, or lotion by formulation with pharmaceuticallyacceptable vehicles administered to a fungally affected area.

EXAMPLES

[0035]FIGS. 1-4 show examples of sites for deuteration of the rapamycinmolecule. Nonlimiting examples of deuterated rapamycin molecules includethe-compounds; 7-deuteromethyl rapamycin (FIG. 1), epi-7-deuteromethylrapamycin (FIG. 2), 7,43-d₆-rapamycin (FIG. 3) and 31,42-d₂-rapamycin(FIG. 4) including the cis and trans isomers of the compounds shown inFIGS. 1-4. FIG. 5 shows the preparation and structure of the compoundglycosylated deuterorapamycin.

Example 1

[0036] Preparation of 7-Deuteromethyl Rapamycin (FIG. 1)

[0037] 5 mg of Rapamycin was dissolved in 2.5 ml of dichlordmethane. 40mg of deuterated methanol was added. 10 beads of NAFION® catalyst wereadded to the above solution. The contents were stirred under nitrogen atroom temperature for 14 hours. The reaction was monitored by massspectrum. The solution was filtered and concentrated The residue wasdissolved in dry; benzene and freeze dried. The white solid obtained washomogenous by mass spectrum analysis and characterized by LC/MS.

Example 2

[0038] Preparation of 31, 42 d₂-7-deuterated Rapamycin (FIG. 3)

[0039] Rapamycin (11 mM) was dissolved in a mixture of cyclohexane anddichloromethane (1:1) 10 ml. The contents were cooled in ice bath andpoly(vinylpyridinium)dichromate 0.5 grams was added. The reactionmixture was stirred overnight and the reaction was followed by massspectrum. The reaction mixture-was filtered, washed with water and driedusing anhydrous magnesium sulphate. The organic solution was filteredand concentrated. The crude product was subjected to purification bysilica column using chloroform-methanol (20:10) mixture. The purefractions were collected and concentrated. The residue was dissolved inbenzene and freeze dried. The product was characterized by LC/MS. M+(Na)932. This material was dissolved in dry ether (10 ml). 10 equivalents oflithium aluminum deuteride was added. The reaction mixture was stirredfor 24 hours. After the completion of the reaction, the excess of LiAlD₄was decomposed by the addition of acetone. The complex was decomposed byadding ice cooled acetic-acid. The mixture is filtered. The filtrate wasdiluted with ether and washed with water, dried, and concentrated. Thecrude mixture was subjected to column chromatography and the requiredmaterial was eluted using chloroform-methanol solvent system. The purefractions were collected and concentrated. The compound was tested bymass spectrum. M=(Na) 940. This compound was converted to the desiredfinal compound (2) by following the procedure as described in Example 1.

Example 3

[0040] Preparation of Glycosylated DeuteroRapamycin (FIG. 5)

[0041] Referring to FIG. 5, compound 10 prepared by example 1 (20 mg)was dissolved in 5 ml of dichloromethane. Dimethylaminopyridine (2.2 mg)was added to the above solution. The contents were cooled to −70 C.4-Nitrophenylchloroformate in dichloromethane was added to the reactionmixture. The solution was stirred under nitrogen at room temperature for14 hours. The reaction was followed by mass spectrum. After thecompletion of the reaction, the reaction mixture was diluted withdichloromethane and the organic solution was washed with water, 0.2M icecold HCl solution. The organic layer was dried over anhydrous magnesiumsulphate. After filtration, the organic solution was filtered andconcentrated. The crude product was purified by LC/MS to provide thepure compound 30 (Yield 10 mg.) Compound 30 (0.9 m.mol)was dissolved indry DMF(0.5 ml) To this mixture, a solution of2-aminoethyl-a-D-glucopyranoside (7.2 m.mol) was added. The reactionmixture was stirred for 14 hours at room temperature. After thecompletion of the reaction, the mixture was diluted withdichloromethane. The organic solution was concentrated in vacuum. Theresidue was extracted with water and the aqueous solution was subjectedto biogel column to get the required pure compound 50. This material wascharacterized by LC/MS. M+(Na)1185.

[0042] Further variations and modifications of the present inventionwill be apparent to those skilled in the art from the foregoing and areintended to be encompassed by the claims appended hereto.

1-32. (cancelled)
 33. A method for the treatment of a disease selectedfrom the group consisting of transplantation rejection, host v. graftdisease, graft v. host disease, leukemnia/lymphoma, hyperproliferativevascular disorder, autoimmune disease, inflammatory disease, solidtumors, and fungal infection, comprising administering to an animal inneed thereof an effective amount of glycosylated deuterorapamycin or apharmaceutically acceptable salt thereof.
 34. The method of claim 33wherein the glycosylated deuterorapamycin is glycosylated at the 42position.
 35. The method of claim 33 wherein the glycosylateddeuterorapamycin has the structure:


36. The method of claim 33 wherein the disease is selected from thegroup consisting of transplantation rejection, host v. graft disease,graft v. host disease, autoimmune disease, and inflammatory disease. 37.The method of claim 33 wherein the disease is selected from the groupconsisting of leukemiallymphoma, hyperproliferative vascular disorder,and solid tumors.
 38. The method of claim 33 wherein the disease is afungal infection.
 39. The method of claim 33 wherein the animal in needis a human.
 40. The method of claim 33 wherein the glycosylateddeuterorapamycin or pharmaceutically acceptable salt thereof isadministered as a pharmaceutical composition comprising the glycosylateddeuterorapamycin or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 41. The method of claim 40 whereinthe pharmaceutically acceptable carrier is selected from the groupconsisting essentially of a solid carrier and a liquid carrier.
 42. Themethod of claim 40 wherein the pharmaceutically acceptable carrier is asolid carrier.
 43. The method of claim 40 wherein the pharmaceuticallyacceptable carrier is a liquid carrier.
 44. The method of claim 40wherein the pharmaceutical composition is in unit dosage form.
 45. Themethod of claim 40 wherein the pharmaceutical composition is in tabletform.
 46. The method of claim 38 wherein the glycosylateddeuterorapamycin or pharmaceutically acceptable salt thereof isadministered as a pharmaceutical composition wherein the glycosylateddeuterorapamycin or pharmaceutically acceptable salt thereof is aformulation selected from the group consisting of a solution, a cream,and a lotion.
 47. The method of claim 33 wherein the glycosylateddeuterorapamyin or pharmaceutically acceptable salt thereof isadministered intramuscularly, intraperitoneally, subcutaneously,intravenously, orally, or topically.